Antioxidant polymer nanocarriers for use in preventing oxidative injury

ABSTRACT

The present invention is a method for encapsulating active protein in a polymeric nanocarrier. The instant method employs homogenization at subzero temperatures so that enzyme activity is retained. Enzymes which can be encapsulated by the present method include, for example, antioxidant enzymes and xenobiotic detoxifying enzymes. Encapsulation of an enzyme protects it from protease degradation and increases therapeutic half-life. Advantageously, polymeric nanoparticles of the invention are permeable to enzyme substrates and therefore enzymes encapsulated by the instant method can exert their effect without release from the nanocarrier. Methods for decomposing a reactive oxygen species, protecting against vascular oxidative stress, and detoxifying a xenobiotic are also provided.

INTRODUCTION

This application claims the benefit of priority of U.S. ProvisionalPatent Application Ser. No. 60/625,727, filed on Nov. 5, 2004, which isincorporated herein in its entirety.

This invention was made in the course of research sponsored by theNational Institutes of Health (NIH Grant No. R01 HL078785-01). The U.S.government may have certain rights to this invention.

BACKGROUND OF THE INVENTION

Oxidative stress induced by reactive oxygen species (ROS) including H₂O₂produced by leukocytes and vascular cells plays a key role inpathogenesis of many disease conditions including atherosclerosis,stroke, hypertension, inflammation, Acute Lung Injury (ALI/ARDS),thrombosis, ischemia-reperfusion injury, organ transplantation,diabetes, angina and myocardial infarction. Therefore, containment ofvascular oxidative stress is important to prophylaxis and treatment ofthese maladies.

Small antioxidants and scavengers can attenuate oxidative stress byterminating lipid peroxidation chain reactions and repairing oxidizedmolecules in the body, yet they are consumed in these reactions and,therefore, are protective only at very high concentrations. Also, theypoorly detoxify directly toxic ROS. Antioxidant inducers, i.e., agentsthat boost production of natural antioxidants and antioxidant enzymes inthe body, also work only at large doses and require pro-longed treatmentto develop protective effects. Therefore, while these antioxidant agentsmay have some utility for alleviating subtle chronic oxidative stress(for example used in form of dietary additions), they have little, ifany value for protection against severe acute insults.

In contrast, antioxidant enzymes (e.g., catalase and superoxidedismutase), are not consumed in reactions with ROS, directly detoxifyROS (this preventing the very initiation of oxidative reactions) and arevery effective even at very low doses. Therefore, antioxidant enzymescan afford more potent protection, which is critically important forcontainment of acute and sub-acute severe oxidative stress, such as thatoccurring in inflammation, stroke, infarction or ALI/ARDS. However,inadequate delivery to endothelial cells lining vascular lumen hashampered their effectiveness for treatment of these and otherpathological conditions involving vascular oxidative stress (Muzykantov(2001) J. Control. Rel. 71:1-21).

In order to improve delivery to endothelium, representing both a sourceof ROS and a critically important, vulnerable target of oxidants(Springer (1990) Scand. J. Immunol. 32:211-216; Varani, et al. (1990)Shock 2:311-319; Heffner & Repine (1989) Am. Rev. Respir. Dis140:531-554), diverse means of delivery have been designed (Kozower, etal. (2003) Nat. Biotechnol. 21:392-398; McCord (2002) Methods Enzymol.349:331-341). For example, targeting of catalase conjugated withantibodies against endothelial cell adhesion molecules ICAM-1 andPECAM-1 boosts vascular antioxidant defense and alleviates oxidativestress in cell cultures (Muzykantov, et al. (1999) Proc. Natl. Acad.Sci. USA 96:2379-2384; Sweitzer, et al. (2003) Free Radic. Biol. Med.23:1035-1046), perfused organs (Atochina, et al. (1998) Am. J. Physiol.275:L806-L817), lung transplantation in rats (Kozower, et al. (2003)supra) and lung injury in mice (Christofidou-Solomidou, et al. (2003)Am. J. Physiol. 285:L283-L292). In addition to enhanced delivery oftherapeutics, targeting cell adhesion molecules inhibits leukocyteadhesion to the endothelium, thus attenuating their pro-inflammatoryfunctions (DeMeester, et al. (1996) Transplantation 62:1477-1485; Lefer,et al. (1996) Am. J. Physiol. 270:H88-H98; Kumasaka, et al. (1996) J.Clin. Invest. 97:2362-2369)

Studies have revealed that enzymes targeted to endothelial cells(including ICAM-1 and PECAM-1 directed conjugates) enter endothelialcells via a novel internalization mechanism, cell adhesionmolecule-mediated endocytosis (Muro et al. (2003) J. Cell. Sci.116:1599-1609), which provides a pathway for intracellular drug deliveryof sub-micron drug-loaded carriers targeted to ICAM-1 or PECAM-1(Wiewrodt, et al. (2002 Blood 99:912-922). This enhances detoxificationof injurious diffusible intracellular oxidants and minimizes catalaseshedding from cell surface (Muro et al. (2003) supra). Using a modelpolystyrene nanoparticle system with surface-absorbed catalase, it wasfound that the subsequent intracellular trafficking led to a lysosomaldestination and degradation of catalase within 3 hours after delivery,restricting the duration of antioxidant protection (Muro, et al. (2003)Am. J. Physiol. Cell Physiol. 285:C1339-C1347). Moreover, othernanoparticle systems are suggested for encapsulation of proteins (see,e.g., U.S. Pat. Nos. 5,543,158 and 6,007,845); however, loadingprotocols for maintaining functional activity of cargo enzymes arelacking.

Accordingly, there is a need in the art for a delivery system fortargeting active therapeutic enzymes and other therapeutic proteins tocells which provides protection of the proteins from subsequent cellulardegradation. The present invention meets this need in the art.Furthermore, it establishes a novel class of drug delivery systems basedon polymer nanocarriers loaded with encapsulated active enzymes that arenot only protected against proteolysis, but capable of carrying outtheir therapeutic function in the body and inside the target cellswithout need for drug release from the carrier, due to detoxification oftoxic compounds (e.g., ROS) diffusing through the polymer carriers.

SUMMARY OF THE INVENTION

The present invention is a method for producing a polymericnanocarrier-encapsulated protein composition resistant to proteasedegradation. The method involves the steps of homogenizing at least oneprotein and an organic polymer solution at subzero temperature so thatan emulsion is formed, mixing the emulsion with an aqueous phase, andhomogenizing the mixture to produce a polymeric nanocarrier-encapsulatedprotein composition. Certain embodiments of the invention provide forencapsulation of antioxidant enzymes and enzymes involved in thedetoxification of xenobiotics. In other embodiments, an affinity moietyis conjugated to the surface of the nanocarrier. In still otherembodiments, that the polymer is selected for pH-dependent degradation.

Polymeric nanocarrier-encapsulated protein compositions are alsoencompassed by the present invention for use in in vivo and in vitromethods for decomposing a reactive oxygen species, protecting againstvascular oxidative stress, and detoxifying xenobiotics.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a nanocarrier system for administration oftherapeutic proteins. It has now been found that homogenization ofproteins with an organic polymer solution at subzero temperatures, andsubsequent homogenization in an aqueous phase, encapsulates the proteinwithin the polymer with concurrent preservation of protein structure andactivity thereby increasing the therapeutic efficacy of the protein. Incontrast to standard double emulsion conditions at 4° C., which yield˜2% loading of proteins into polymeric nanoparticles, subzerohomogenization enhances loading by ˜10-fold. Not wishing to be bound bytheory, it is believed that by incorporating a freeze-thaw cycle duringpolymeric nanocarrier synthesis, the polymer phase precipitates aroundthe primary emulsion, improving overall encapsulation. This is supportedby the observation that at higher polymer concentrations (>100 mg/mL)the freeze-thaw cycle results in the complete precipitation of thepolymer phase.

A protein, as used in the context of the present invention refers to amolecule composed of amino acids joined by peptide linkages. Includedwithin the term protein are structural proteins such as albumins,globulins, histones, collagens, elastins, and keratins; and proteinswith a chemical function to fulfill, e.g., enzymes. Also included areprotein molecules united with nonprotein molecules to produce compoundproteins such as nucleoproteins, mucoproteins, lipoproteins andmetalloproteins. A protein of the invention can be naturally-occurring,synthetic or semi-synthetic.

Functionally active proteins that are particularly useful forencapsulation in the instant polymeric nanocarrier include clinicallyrelevant proteases and their inhibitors such as serpins (Schimmoller, etal. (2002) Curr. Pharm. Des. 8:2521-31; Rosenblum & Kozarich (2003)Curr. Opin. Chem. Biol. 7:496-504; Barnes & Hansel (2003) Lancet364:985-96); growth factors and hormones (Bremer, et al. (1997) Pharm.Biotechnol. 10:239-54; Rosier, et al. (1998) Clin. Orthop. S294-300;Chen & Mooney (2003) Pharm. Res. 20:1103-12; Peppas, et al. (2004)Expert Opin. Biol. Ther. 4:881-7); enzymes, e.g., for replacementtherapies (Layer, et al. (2001) Curr. Gastroenterol. Rep. 3:101-8;Meikle & Hopwood (2003) Eur. J. Pediatr. 162(Suppl 1):S34-7; Mignani &Cagnoli (2004) J. Nephrol. 17:354-63); anticoagulants and fibrinolyticplasminogen activators (Harker, et al. (1997) Thromb. Haemost.78:736-41; Wieland, et al. (2003) Curr. Opin. Investig. Drugs 4:264-71);interferons and cytokines (Burke (1999) Cytokines Cell. Mol. Ther.5:51-61; Younes & Amsden (2002) J. Pharm. Sci. 91:2-17; Barnes (2003)Cytokine Growth Factor Rev. 14:511-22); as well as an antibodies,antibody fragments and their conjugates with toxins and otherbiologically active agents (Foster (1996) J. Allergy Clin. Immunol.98:S270-7; Muzykantov (2001) J. Control. Rel. 71:1-21; Thorpe (2004)Clin. Cancer Res. 10:415-27).

In one embodiment, the polymeric nanocarrier-encapsulated protein is anantioxidant enzyme which is capable of reducing oxidative damage bydecomposing or degrading reactive oxygen species. Antioxidant enzymesparticularly useful include: catalase, glutathione peroxidase,superoxide dismutase, hemeoxygenase, glutathione-S-transferase, orsynthetic or mimetic enzymes thereof. An antioxidant enzyme encapsulatedin the instant polymeric nanocarrier is particularly useful in methodsfor detoxifying reactive oxygen species including the superoxide anionradical (O_(2.) ⁻), hydrogen peroxide (H₂O₂), hypochlorous acid (HOCl),hydroxyl radical (OH.), and singlet oxygen (¹O₂) which are generated inthe body, mediating cell damage and apoptosis. When the instantpolymeric nanocarrier contains an antioxidant enzyme and an affinitymoiety for targeting vascular endothelial cells, sustained therapyagainst vascular oxidative stress can be achieved for the prevention ortreatment of pathological processes involved in disease conditionsincluding atherosclerosis, hypertension, diabetes, stroke, myocardialinfarction, acute lung injury, inflammation and ischemia-reperfusioninjury.

In another embodiment, the polymeric nanocarrier-encapsulated protein isan enzyme which detoxifies one or more xenobiotics. In accordance withits classical definition, a xenobiotic is defined as a compound ormolecule which is foreign to the body or a living organism. As such, axenobiotic is intended to include insecticides, elicit drugs,pharmaceutical agents, organic chemicals, chemical warfare agents,toxins (including endotoxins), and the like which can have an adverseeffect on a subject. Enzymes which detoxify xenobiotics can be used toreduce, inhibit, or ameliorate the effects of an intentional orunintentional exposure (including overdosing) to one or morexenobiotics. Moreover, detoxifying enzymes can be provided to subjectswith impaired liver function, e.g., due to alcoholism, fatty liverdisease, biliary cirrhosis, and hepatocarinomas leading to lowerdetoxification activity in general (Lee (1995) N. Engl. J. Med.333:1118-1127), or suffering from a peroxisomal disorder such ashyperoxaluria, Refsum disease, and β-Oxidation disorders. Xenobioticdetoxifying enzymes particularly suitable for encapsulation in theinstant polymeric nanocarrier include, but are not limited to,cytochrome P450 enzymes such as Cyp3A4 and Cyp3A5, Cyp1A1, Cyp1A2,Cyp2D6, Cyp2E1, Cyp2C, Cyp2C9, Cyp2B6, Cyp2C19 and the like which areresponsible for the metabolism of a variety of drugs includingcyclosporin, nifedipine, warfarin, phenacetin, caffeine, aflatoxin B1,ethanol, carbon tetrachloride, coumarin, sparteine, cyclophosfamide,etc. (Iarbovici (1997) J. NIH Res. 9:34-45; Benet, et al. (1996) In: ThePharmacological Basis of Therapeutics, Molinoff, et al. (eds.), 9^(th)edition new York, NY: McGraw-Hill, pp 3-27; Vermeulen (1996) In:Cytochrome P450: Metabolic and Toxicological Aspects, Ioannides, ed.,Boca Raton, Fla.: CRC Press, Inc. pp 29-53); alcohol dehydrogenase;epoxide hydrolase; glucuronyl transferases (detoxifying phenols, thiols,amines, and carboxylic acids); sulfotransferase (detoxifying phenols,thiols, and amines); N- and O-methyl transferases (detoxifying phenolsand amines); N-acetyl transferase (detoxifying amines); and otherperoxisomal enzymes including peroxidases, catalase, phytanoyl-CoAhydroxylase, and α-methylacyl-CoA racemase. In cases where thexenobiotic is of an unknown origin, it is contemplated that a polymericnanoparticle containing a plurality of detoxifying enzymes can beemployed to facilitate detoxification of the unknown agent.

Accordingly, while particular embodiments of the present inventionembrace the encapsulation of at least one protein in a polymericnanocarrier of the invention, other embodiments provide that at leasttwo, three, four, five, or more types of proteins are encapsulated inthe instant polymeric nanocarrier.

Advantageously, the proteins encapsulated in the instant polymericnanocarriers can exert their effect without release from the polymericnanocarriers. Therefore, in particular embodiments of the presentinvention, the protein encapsulated in the instant polymeric nanocarriercomposition is not released from the polymeric nanocarrier during thecourse of use due to direct diffusion of substrates (such as toxiccompounds including ROS) through the polymeric matrix of the carrier anddecomposition within the nanocarrier. In other embodiments, however,gradual degradation of the polymer carrier (e.g., in a pH-dependentmanner) can be used for controlled-release of encapsulated cargo(s)thereby regulating the extent and duration of the activity of the cargo.

In accordance with the instant method, the protein and an organicpolymer solution are homogenized to form an emulsion. Homogenization isintended to mean a mechanical process for reducing the size of a organicpolymer particle of an emulsion to uniform size. Homogenization stepscan be carried out using any conventional ultrasound or homogenizer inaccordance with the teachings disclosed herein, wherein rate and time ofhomogenization can vary depending upon the polymeric nanocarriercharacteristics desired. Particularly suitable homogenization parametersfor the first homogenization step of the instant method include a ratein the range of 5 to 20 krpm, or more desirably 9 to 16 krpm, for lessthan one minute. Particularly suitable homogenization parameters for thesecond homogenization step of the instant method include a rate ofhomogenization in the range of 5 to 20 krpm, or more desirably at least15 krpm, for less than one, two, three or four minutes. Alternatively,pressurized homogenization strategies can be used in conjunction withthe freeze-thaw encapsulation process.

While a standard double-emulsion method carried out at 4° C. provides˜2% loading of a protein in a polymeric nanocarrier, particularembodiments of the present invention embrace carrying out the firsthomogenization step of the instant method at subzero temperature. Asused herein, subzero temperature is intended to encompass a temperaturein the range of −180° C. to 0° C. In particular embodiments, subzerotemperatures are in the range of −40° C. to −100° C. The particulartemperature selected can be dependent upon the melting point of thesolvent used and the protein being encapsulated. For example, a subzerotemperature of greater than −97° C. is desired when dichloromethane isused. Similarly, temperatures greater than −67° C., −87° C., −78° C. or−97° C. should be used when chloroform, ethyl acetate, butyl acetate, oracetone solvents are respectively employed.

A solvent of the present invention is used for suspending the instantpolymer in solution, wherein the particular solvent selected can be anaqueous solvent or an organic solvent. Suitable solvents of the presentinvention include alkylated alcohols, ethers, acetone, alkanes, dimethylsulfoxide, toluene, cyclic hydrocarbons, benzene, and the like.

The term polymer or polymeric refers to molecules formed from thechemical union of two or more repeating units. Accordingly, includedwithin the term polymer may be, for example, dimers, trimers andoligomers. The polymer can be synthetic, naturally-occurring orsemi-synthetic. In a particular form, polymer refers to molecules whichare 10 or more repeating units.

In certain embodiments, the organic polymer of the present invention isbiodegradable. In other embodiments, the organic polymer is a blockcopolymer, i.e., a combination of two or more chains of constitutionallyor configurationally different features. Block copolymers includediblock, triblock, or multiblock copolymers. Examples of biocompatibleorganic polymers suitable for use in block copolymers of the presentinvention are poly(ethylene-covinyl acetate), and silicone rubbercross-linked to poly (dimethyl siloxan sulfoxide) and derivativesthereof, polylactic acid, polyglycolic acid or polycaprolactone andtheir associated copolymers, e.g., poly(lactide-co-glycolide) at alllactide to glycolide ratios, and both L-lactide or D,L-lactide. Inparticular embodiments, a poly(lactic-co-glycolic)acid (PLGA) isemployed. Amphiphilic diblock copolymers composed of hydrophilic blocks,e.g., including polypyrrolidone, poly(amino acids), polyether,polysaccharide or polyacrylic acid and its hydrophilic esterderivatives; and hydrophobic blocks, e.g., polyanhydrides,polydioxanones, polyphosphazenes, polyesters, polylactones,polyfumarates, polymers of alpha-hydroxy carboxylic acids,polyhydroxybutyric acid, polyorthoesters, polycaprolactone,polyphosphates, or copolymers prepared from the monomers of thesepolymers can be used to form copolymers for use in preparing the instantnanocarriers. In one embodiment, the hydrophilic block of the copolymerexists as an ester end-capped form. In another embodiment, thehydrophilic block of the copolymer exists in its native form providinglinkage sites for an affinity moiety. In certain embodiments, thehydrophilic domain of the block copolymer has a molecular weight in therange of 100 to 20000 Daltons. In other embodiments, the hydrophobicdomain of the block copolymer has a molecular weight in the range of2000 to 300000 Daltons. In particular embodiments, the weight fractionof hydrophilic domain does not exceed 75 weight % of the polymercontent. The variety of materials that can be used to prepare the blockcopolymers forming the nanocarriers significantly increases thediversity of protein retention time within the nanocarrier anddegradation profile that can be accomplished in vivo.

Degradation of the hydrophobic-block of the copolymers occurs through ahydrolytic reaction that is acid/base catalyzed. Based on the assaysdisclosed herein, nanocarrier life-spans are greatly reduced underacidic conditions, but can be predetermined by blending large and smallmolecular weight fractions of the diblock copolymer. Due to thisaccelerated rate and the a priori knowledge of lysosomal pH (pH4.5-5.5), the instant nanocarriers can be designed to have pH-dependentdegradation such that their degradation time in the lysosomalcompartments can be regulated to a desirable rate thereby facilitatingrelease of a drug, its longevity in the target cells, and rate ofmetabolization of the whole nanocarrier.

The block copolymers of the present invention are desirably composed ofa polymeric-backbone having functional (e.g., pendant side chain orendcapped) groups for physically cross-linking with other entities,including affinity moieties, therapeutic entities, or other polymers.Functional groups encompass conjugatable groups such as amines,hydroxyls, carbonyls, thiols, and carboxylic acids for covalentlybonding of other bioactive molecules to the surface of the polymericnanocarrier. The linkages formed following conjugation of the bioactivemolecules to the conjugatable groups include amides, esters, andthioethers. Examples of polymers which have conjugatable functionalgroups include (poly)lysine, acetylated poly(lysine), poly(glutamicacid), and polyethylene glycol (PEG) and the like. In particularembodiments, a block copolymer of the present invention containspolyethylene glycol (PEG). Generally PEG polymers for use herein have amolecular weight of from about 1000 to about 7500, or more suitably withmolecular weights of from about 3000 to about 6000.

The emulsion (i.e., mixture of two or more generally immiscible liquids)produced after the first homogenization step of the instant method issubsequently mixed with an aqueous phase at a temperature of 4° C. to25° C. and subjected to a second homogenization to generate the instantpolymeric nanocarrier. A suitable aqueous phase can include water,saline and the like. In particular embodiments the aqueous phasecontains a surfactant. A surfactant refers to a substance that altersenergy relationship at interfaces (e.g., that of organic polymersdisplaying surface activity) and generally encompasses wetting agents,detergents, penetrants, spreaders, dispersing agents, and foamingagents. Surfactants can be of natural, semi-synthetic (modified natural)or synthetic origin. Exemplary natural polymers include naturallyoccurring polysaccharides and proteins, such as albumin. Exemplarysemi-synthetic polymers include carboxymethylcellulose,hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose,and methoxycellulose. Exemplary synthetic polymers includepolyphosphazenes, polyethylenes including PLURONIC® compounds,polypropylenes, polyurethanes such as polyvinyl alcohol andpolyvinylpyrrolidone, and polyamides including polylactic acids. Methodsfor preparing polymeric nanocarriers which employ surfactants will bereadily apparent to those skilled in the art, in view of the presentdisclosure, when coupled with information known in the art (e.g., U.S.Pat. No. 5,205,290).

In particular embodiments, the instant nanocarrier further contains anaffinity moiety. An affinity moiety refers to any material or substancewhich can promote targeting of the compositions of the present inventionto particular cells, tissues and/or receptors in vivo or in vitro. Theaffinity moiety can be synthetic, semi-synthetic, ornaturally-occurring. Materials or substances which can serve as affinitymoieties include, for example, proteins, including antibodies, antibodyfragments, hormones, hormone analogues, glycoproteins and lectins,peptides, polypeptides, amino acids, sugars, saccharides, includingmonosaccharides and polysaccharides, carbohydrates, vitamins, steroids,steroid analogs, hormones, cofactors, bioactive agents, and geneticmaterial, including nucleosides, nucleotides, nucleotide acid constructsand polynucleotides. Particularly suitable affinity moieties includemolecules which specifically bind to receptors or antigens found onvascular cells. Other suitable affinity moieties target endothelialreceptors, tissues or other targets accessible through a body fluid orreceptors or other targets upregulated in a tissue or cell adjacent toor in a bodily fluid. For example, affinity moieties attached tonanocarriers designed to deliver proteins to the eye can be injectedinto the vitreous, choroid, or sclera; affinity moieties attached tonanocarriers designed to deliver proteins to the joint can be injectedinto the synovial fluid; or affinity moieties to the spine and brain canbe delivered into the cerebral spinal fluid.

The affinity moiety can have other effects, including therapeuticeffects, in addition to specifically binding to a target. For example,the affinity moiety can modulate the function of an enzyme target. Bymodulating cellular function, the affinity moiety is meant toalter/enhance cellular response when compared to not adding the affinitymoiety. In most cases, a desired form of modulation of function isinhibition. Examples of affinity moieties which can have other functionsor effects include agents such as Combrestastatin A4 Prodrug (CA4P)which can be used as a vascular affinity moiety that also acts as ananti-angiogenesis agent; and Cidecin, a cyclic lipopeptide, used as abactericidal and anti-inflammatory agent.

Exemplary affinity moieties attached to the polymeric nanocarrier of thepresent invention include, but are not limited to, peptides such asRGD-containing peptides (e.g. those described in U.S. Pat. No.5,866,540); bombesin or gastrin-releasing peptide; antibodies such asanti-PECAM; and peptides designed de novo to be complementary totumor-expressed receptors, antigenic determinants, or other receptortargeting groups. These affinity moieties can be used to control thebiodistribution, non-specific adhesion, and blood pool half-life of thepolymeric nanocarrier compositions. In particular embodiments, theaffinity moiety is attached by covalent means. In another embodiment,the attachment is by non-covalent means. For example, antibody affinitymoieties can be attached by a biotin-avidin biotinylated antibodysandwich to allow a variety of commercially available biotinylatedantibodies to be used on the coated polymeric nanocarrier. In otherembodiments, the affinity moiety is added in a single step, e.g.,through the coupling of biotinylated nanocarriers andantibody-streptavidin chemical conjugate or fusion construct.

The size of the instant nanocarrier can be adjusted for the particularintended end use including, for example, environmental detoxification ortherapeutic use. As the size, feed ratio, and homogenization conditionsof polymer can be readily manipulated, the overall size of thenanocarrier can be adapted for optimum passage of the nanocarrierthrough the permeable vasculature at the site of pathology, as long asthe agent retains sufficient size to maintain its desired properties(e.g., circulation life-time). Accordingly, the nanocarriers of thepresent invention can be sized at any given diameter within the intervalbetween 20 and 20,000 nm as desired. In addition, the size of thenanocarrier can be chosen so as to permit a first administration ofnanocarrier of a size that cannot pass through the permeablevasculature, followed by one or more additional administrations ofnanocarriers of a size that can pass through the permeable vasculature.In connection with particular uses, for example, intravascular use, itmay be desirable that the vesicles be no larger than about 500 nm indiameter, with smaller vesicles being most desired. In certainembodiments of the present invention, a polymeric nanocarrier of thepresent invention has a diameter of less than 1 micron. In otherembodiments, a 50 to 900 nm polymeric nanocarrier is produced. Inparticular embodiments, the polymeric nanocarrier is in the range ofapproximately (i.e., ±50 nm) 100 nm to 400 nm in diameter.

The present polymeric nanocarrier compositions are desirably formulatedin an aqueous environment. Diluents which can be employed to create suchan aqueous environment include, for example, normal saline andphysiological saline, water, including deionized water or watercontaining one or more dissolved solutes, such as salts or sugars, whichpreferably do not interfere with the formation and/or stability of thenanocarrier or their therapeutic use.

To illustrate the preparation and use of the instant polymericnanocarrier compositions, PEG-PLGA nanocarrier-encapsulated catalase wasprepared and used to degrade the reactive oxygen species, hydrogenperoxide. To achieve targeted vascular antioxidant therapy, polymericnanocarriers can contain functional groups for the surface attachment ofaffinity moieties such as antibodies to endothelial adhesion molecules(Muzykantov, et al. (1999) supra; Sweitzer, et al. (2003) surpa;Atochina, et al. (1998) supra; Christofidou-Solomidou, et al. (2003)supra; DeMeester, et al. (1996) supra). Thus, a PEG moiety was selectedfor facilitating surface attachment (Mercadal, et al. (2000) Biochim.Biophys. Acta 1509:299-310; Maruyama, et al. (1997) Adv. Drug Deliv.Rev. 24:235-242; Olivier, et al. (2002) Pharm. Res. 19:1137-1143). Inaddition, external PEG chains impart stealth properties to polymericnanocarriers (Mosqueira, et al. (2001) Pharma. Res. 18:1411-1419;Photos, et al. (2003) J. Control Rel. 90:323-334). Conjugation wascarried out using copolymers of 38 kD PLGA and 10 kD PEG with anunblocked terminal hydroxyl group. PEG and lactic acid assays showedthat the polymer contained 11 weight % PEG (50% conjugation yield). Gelpermeation chromatography confirmed that the molecular weight was 50,000with a polydispersity index (PDI) of 2.03. Fourier transform infraredspectroscopy (FTIR) analysis verified presence of both the carbonhydrogen stretch of PEG saturated backbone at 2850 cm⁻¹ and the esterpeak of the PLGA at 1790 cm⁻¹ in the copolymer.

To demonstrate that catalase, residing inside the nanocarrier, coulddegrade H₂O₂ which permeates across the nanocarrier barrier, thediffusivity of hydrogen peroxide through PLGA was measured. Becausediffusivity is a bulk property, it is insensitive to geometry. Thus, aclassical two-chamber polymer-film diffusion study was employed. Fromthese experiments, it was demonstrated that H₂O₂ could easily diffusethrough the PLGA polymer (film thickness varied from 80 to 200 μm).Under the experimental conditions, steady-state and a constant drivingforce was assumed. The following equation (1), derived from Fick's firstlaw of diffusion, was used to calculate the film permeability (Bell &Peppas (1996) Biomaterials 17:1203-1218).

$\begin{matrix}{{\ln( {1 - \frac{2C_{t}}{C_{o}}} )} = {{- \frac{2A}{V}}\frac{Dk}{1}t}} & (1)\end{matrix}$where A is the area of the diffusion plane, V is the volume of thereceptor cell, D the diffusivity, k the partition coefficient, 1 thethickness of the polymer film, C_(o) the concentration of the donor celland C_(t) the calculated cumulative concentration of the receptor cellat time, t. Averaging data of two PLGA films with three replicates each,diffusivity of H₂O₂ through PLGA was found to be 3.3±0.37×10⁻⁷ cm²/s.While diffusion of H₂O₂ through PLGA polymer was slower than in water,the rate was 10 times faster than with lipid membranes (Seaver & Imlay(2001) J. Bacteriol. 183:7182-7189).

The synthesis of polymeric nanoparticles consisting of PEG-PLGA andother biodegradable polymers provides examples of polymeric nanoparticleloading with small soluble drugs resistant to harsh conditions ofpolymeric nanoparticle formulation (Avgoustakis, et al. (2002) J.Control. Rel. 79:123-135; Suh, et al. (1998) J. Biomed. Mater. Res.42:331-338; DeCampos, et al. (2001) Int. J. Pharma. 224:159-168).However, loading of enzymes into a polymeric matrix under such harshconditions usually inactivates the enzymes. For example, a probesonication, double emulsion followed by rapid solvent evaporationemployed for encapsulation of L-aspariginase and interferon-alphaimpaired the activity of both proteins (Sanchez, et al. (2003) Eur. J.Pharm. Sci. 18:221-229; Gaspar, et al. (1998) J. Control. Rel.52:53-62). Loading of protein C (a 60-kD monomer serine protease thatcleaves coagulation factors) into mPEG-PLGA polymeric nanoparticlesusing a similar probe sonication approach using an acetone/DCM mixturecaused protein C inactivation, yet a fraction of released protein Ccould be recovered (Zambaux, et al. (2001) Int. J. Pharm. 212:1-9).

Likewise, in the presence of DCM, catalase activity was reduced by 80%after a 10 second of sonication, while the acetone/DCM (50:50)co-solvent mixture actually exacerbated inactivation, with 60% activityloss even without sonication (Table 1). These data indicate lowendurance of large multimeric enzymes to loading, which likely dependson complex cargo quaternary structure (catalase is a 240-kD tetramercontaining central coordinated heme redox group). Since thissonication-induced deactivation was not observed in a pure aqueoussystem, an interfacial-mediated unfolding of catalase is the likelymechanism of deactivation.

TABLE 1 Percent Activity Remaining Homogenization (20 krpm) DCM withoutSonication (20 W) Time (seconds) Freezing DCM at −80° C. DCM Acetone/DCM0  100 ± 8.73 90.10 ± 9.92 100.00 ± 13.00  37.91 ± 4.41 5 — — 63.75 ±16.28 51.04 ± 7.82 10 80.21 ± 10.16 61.64 ± 6.72 22.61 ± 6.57  28.11 ±2.80 20 — — 15.29 ± 11.27  1.85 ± 1.08 30 104.71 ± 8.66  44.20 ± 4.73 —— 60 87.84 ± 8.30  56.46 ± 8.28 — —

To load catalase into PEG-PLGA matrix during polymeric nonocarrierformation without loss of enzymatic activity, a double emulsion-solventevaporation method (Avgoustakis, et al. (2002) supra; Zambaux, et al.(2001) Int. J. Pharm. 212:1-9), employing either ultrasound ormechanical homogenization for emulsification, was used. When the latterapproach was employed, catalase retained ˜90% activity after 1 minute15000 RPM homogenization in acetone/DCM at 4° C. and ˜60% when a ˜80° C.freeze-thaw cycle was included. Since mechanical emulsification producesparticles with a wide size-distribution, serial centrifugations andfiltration through a one-micron filter were employed to isolate thepolymeric nanocarrier fraction (particles<700 nm diameter) from largermicroparticles. Double emulsion polymeric nanocarriers possess both aninner and external aqueous phase, resulting in an enhanced energypenalty that makes the loading of aqueous drug inside polymericnanocarrier pockets unlikely. Since entropy works against loading,conceivably the polymeric nanocarrier loading is not determined byequilibrium partition of the drug into the polymeric nanocarrier, butrather by the kinetic effects of polymer gelation that reduces the inneraqueous domain release into the outer aqueous compartment. As such,standard loading conditions resulted in poor loading (2.25% ±0.82% of¹²⁵I-catalase). However, when polymer precipitation/gelation was inducedby a freeze-thaw cycle in the primary emulsion step, catalase loadinginto the polymeric nanocarrier fraction was enhanced to a loadingefficiency of 13.5% ±2.95%.

As particle size decreases, the interface between the oil and waterphase increase. To initially overcome the energy barrier, energy (e.g.,mechanical homogenization) is added to the system. However, once energyinput is ceased, the oil phase starts to coalesce into increasinglylarger sizes unless enough surfactant is present to stabilize thesystem, or the polymer solidifies prior to coalescence. This hardeningof the polymer phase is controlled predominately by the solventevaporation rate (a kinetic parameter) but is also determined by thepolymer molecular weight and intrinsic features of the solvent. Inagreement with this, size of the polymeric nanocarriers decreased withincreasing energy input in the secondary emulsion down to a minimum sizedetermined by the surfactant load of the system (Table 2).

TABLE 2 2^(nd) Homogenization Size (nm ± SE) Rate (krpm) 11 Weight % PEG5 Weight % PEG 5 292.6 ± 7.3   733.3 ± 111.0 10 277.8 ± 13.7 497.2 ±72.4 13.5 270.7 ± 13.1 534.2 ± 92.7 15  256 ± 5.8 367.2 ± 40.0 20 —359.5 ± 20.0

Determining polymeric nanocarrier size as a function of rate²/time, ascaling factor for energy input, illustrates his effect (Table 3).

TABLE 3 Energy Input Size (nm ± SE) (krpm² * time) 11 Weight % PEG 5Weight % PEG 25 326.6 ± 16.0 692.3 ± 98.3 100 309.0 ± 17.8 545.3 ± 46.2112.5 319.8 ± 9.7  — 182.25 — 547.6 ± 46.5 225 297.6 ± 7.3  416.6 ± 31.1400 291.4 ± 9.9  300.2 ± 8.9  1125 280.6 ± 10.1 —

The average polymeric nanocarrier size decreased with an increase inhomogenization rate and time (Table 2, Table 4).

TABLE 4 2^(nd) Homogenization Time (Minutes) Size (nm ± SE) 0.5 337.2 ±14.9 1.0 275.6 ± 32.2 2.0 291.4 ± 17.1 5.0 280.6 ± 17.6

Polymeric nanocarriers decreased in size from 350 to 250 nm as thehomogenization rate increased from 5 to 20 krpm. When the PEG content inthe PEG-PLGA was decreased from 11 to 5 weight %, the size dependencebecame even more evident, varying from 700 to 350 nm. To analyzesignificant number of samples and estimate reproducibility, dynamiclight scattering measurements were used to determine polymericnanocarrier size in most experiments. Electron microscopy confirmed sizeof polymeric nanocarriers determined by dynamic light scattering.

To assess the role of surfactant stabilization in polymeric nanocarriersynthesis, the effect of PVA surfactant was analyzed for PEG-PLGApolymeric nanocarriers with 5 weight % PEG. As PVA concentrationincreased (from 0.1 to 4 weight %), the size of the polymericnanocarrier increased for both second homogenization rates tested, 15and 20 krpm (Table 5). For each equivalent PVA concentration, thepolymeric nanocarrier formed at 20 krpm was smaller than the 15 krpmcounterpart.

TABLE 5 PVA Concentration Particle Size (nm ± SE) (weight %) 15 krpm 20krpm 0.1 296.0 ± 8.5 267.8 ± 2.4 1.0 295.4 ± 3.4 277.2 ± 6.9 2.0 320.9 ±6.6 302.4 ± 4.7 4.0  372.0 ± 41.8  359.5 ± 20.0

Increased surfactant concentration also increased the polymericnanocarrier yield (5 to 25 weight %; Table 6).

TABLE 6 PVA Concentration Nanoparticle yield (weight %) (% ± SE) 0.1 4.3 ± 1.2 1.0  6.2 ± 1.9 2.0 10.8 ± 1.5 4.0 24.7 ± 9.9

The interfacial area of the oil-to-water phase was defined by thefollowing equation (2):

$\begin{matrix}{{IA} = {{C_{particles}{SA}_{{particles} =}} = \frac{3C_{mass}}{\rho\; r}}} & (2)\end{matrix}$where C_(particles) is the number concentration of polymericnanocarrier, SA_(particle) is the surface area of the polymericnanocarrier, C_(mass) is the mass concentration of the particles, ρ isthe polymer density (assumed to be 1.2 g/cm³), and r is the meanparticle radius. This analysis showed an overall increase in oil/watersurface area per volume of emulsion (Table 7).

TABLE 7 Interfacial PVA Concentration Area/Volume Emulsion (weight %)(1/cm ± SE) 0.1 18.7 ± 1.9 1.0 16.0 ± 5.1 2.0 41.6 ± 3.5 4.0  80.1 ±15.8

Results of chemical assays of the conjugated diblock PEG-PLGA confirmedthe conjugation efficiency to be 50%. This result represents arelatively under-appreciated aspect of polymeric nanocarrier synthesis,i.e., role of diblock PEG-PLGA to monoblock PLGA feed ratio. Themajority of polymeric nanocarrier prepared herein was synthesized usingeither PEG-PLGA in its pure form or mixed with bulk PLGA. It was foundthat the diblock PEG-PLGA to monoblock PLGA feed ratio represented asignificant factor that controls size of the resultant polymer particles(Table 8)

TABLE 8 PEG-PLGA Constant PEG-PLGA (constant weight % PEG) (10 mg/mL) +PLGA Interfacial Interfacial PEG-PLGA Particle Area/Volume ParticleArea/Volume Concentration Size Emulsion Size Emulsion (mg/mL) (nm ± SE)(1/cm ± SE) (nm ± SE) (1/cm ± SE) 5 412.3 ± 8.7  6.5 ± 0.7 — — 10 349.7± 4.6  9.2 ± 1.3 422.0 ± 36.6 12.1 ± 3.5 25 306.5 ± 7.9 21.2 ± 1.7 467.2± 59.7 14.5 ± 2.0 50  280.0 ± 14.9 21.7 ± 2.4 563.2 ± 22.5 16.9 ± 4.8100 263.5 ± 6.5 54.0 ± 2.0  692.3 ± 106.8  5.0 ± 0.7

Increases in PEG-PLGA diblock content decreased the subsequent polymericnanocarrier size, possibly due to the surfactant qualities afforded bythis amphiphile. This is further substantiated by the fact thatincreasing polymer concentration with a constant weight % of PEG (5weight %) resulted in a decrease in polymeric nanocarrier size (from 400to 250 nm) and an increase in polymeric nanocarrier yield (from 1 to 5mg). In contrast, when the polymer concentration was increased with aconstant 10 mg/mL PEG-PLGA concentration, particle size increased from400 to 700 nm and the interfacial area per volume remained relativelyconstant. Moreover, increases in PEG-PLGA concentration increased yield(Table 9). The measured PEG content of the polymeric nanocarrier matchedthat of the feed conditions, confirming that PEG-PLGA did notpreferentially self-associate into micelle structures and polymericnanocarrier polymer composition can be predetermined by simply alteringthe copolymer mixtures.

TABLE 9 PEG-PLGA Concentration (mg/mL) Nanocarrier Yield (% ± SE) 5 0.8± 0.1 10 1.0 ± 0.1 25 2.1 ± 0.1 50 1.8 ± 0.2 100 4.3 ± 0.1

The activity of loaded catalase was determined by the direct monitoringA₂₄₂ nm absorbance of H₂O₂ that is relatively stable in water in minutetime scale. The addition of unloaded polymeric nanocarrier elevated anet absorbance due to light scattering, but produced no measurablesubsequent decrease in H₂O₂. In a sharp contrast, catalase-loadedpolymeric nanocarriers produced the same increase in absorbance that wasimmediately followed by a decrease in H₂O₂ absorbance, compatible to thekinetics of a similar amount of free catalase. Therefore, catalaseloaded into polymeric nanocarriers retained its enzymatic activity andeffectively decomposed H₂O₂ diffusing through the polymer shell.

Increases in the rate of homogenization in the primary emulsion (up to20 krpm) did not alter catalase activity, but a primary homogenizationtime extending beyond 1 minute at 15 krpm inactivated catalase-loadedpolymeric nanocarrier (Table 10). Because the first emulsionhomogenization has limited effect on polymeric nanocarrier size, a1-minute homogenization is sufficient for the effective synthesis ofpolymeric nanocarrier in the size range of interest.

TABLE 10 Enzyme Activity (Units/mg polymeric Time (minutes) nanocarrier± SE) 0.25 3.3 ± 0.4 0.75 3.2 ± 1.8 2.00 0.1 ± 0.1 5.00 0.06 ± 0.04

Loading of catalase into the primary emulsion without versus with afreeze-thaw cycle endowed the polymeric 5 nanocarriers with marginalversus substantial catalase activity, respectively, under the secondaryhomogenization rates analyzed (Table 11). This was in correlation with¹²⁵I-catalase loading data.

TABLE 11 Enzyme Activity Units/mg polymeric 2^(nd) Homogenizationnanocarrier ± SE) Speed (krpm) Freeze-Thaw No Freeze-Thaw 5.0 0.97 ±0.06 0.09 ± 0.07 10.0 2.07 ± 0.87 0.13 ± 0.04 13.5 — 0.19 ± 0.07 15.02.50 ± 0.74 0.12 ± 0.06 20.0 1.29 ± 0.28 —

Second emulsion homogenization times longer than one minute alsodeteriorated catalase activity (0.91±0.36 U/mg polymeric nanocarrierwith 1-minute homogenization versus 0.42±0.06 U/mg polymeric nanocarrierwith 5 minutes homogenization). The homogenization rate of the secondemulsion displayed a bell-shape optimum of catalase-loaded polymericnanocarrier activity (˜15 krpm) followed by reduction at 20 krpm.

Supporting this result; ¹²⁵I-catalase loading into polymeric nanocarrierwas >20% versus <5% at 15 krpm versus 25 krpm, respectively, in secondemulsion. Unexpectedly, at the polymer concentration used (25 mg/mL),the loading efficiency was independent of polymer composition andmolecular weight (Table 12). Thus, polymer molecular weight andcomposition can be used to control polymeric nanocarrier properties suchas degradation rate, sizing, and in vivo circulation.

TABLE 12 Loading Efficiency (% ± SE) Polymer Size 15 krpm 25 krpmmPEG-PLA 50 kDa 12.8 ± 2.1 4.6 ± 2.4 30 kDa 21.9 ± 7.1 4.2 ± 3.3 20 kDa15.2 ± 6.5 4.3 ± 4.0 PEG-PLGA 50 kDa 13.5 ± 5.2 2.4 ± 1.7

In addition to compositional analysis, it was determined whethernanocarriers protect loaded catalase from proteolysis. After a 4-hourincubation with PRONASE® free catalase possessed <1% of initialH₂O₂-degrading activity, while catalase-loaded polymeric nanocarrierretained 35% of initial activity. Co-incubation of catalase withunloaded polymeric nanocarrier did not protect catalase from PRONASE®,indicating that only catalase encapsulated into polymeric nanocarriersis protected against proteolysis and therefore capable of degrading H₂O₂diffusing through the polymer shell. The time-course of the proteolyticloss of catalase activity was also determined (Table 13). About 90% offree catalase activity was lost after a 1-hour incubation, and fellbelow measurable levels at 6 hours. In contrast, catalase loaded intothe disclosed polymeric nanocarrier formulation retained 40% of itsinitial activity at 6 hours. The activity seemed to reach a plateau with25% of the initial activity remaining at stable level for at least ˜20hours. The loss of activity in the polymeric nanocarrier was believed tobe associated with the catalase that was either surface bound or wasreleased into the aqueous medium. However, the stable, active fractionrepresents catalase residing inside the protease inaccessible domains ofthe polymeric nanocarrier. When the catalase was loaded into polymericnanocarriers formed using a 20% aqueous content in the primary emulsion(instead of the 10 weight % employed through these studies), initialrate of loss of catalase was significantly accelerated. However, after 1hour, the activity stabilized at level of ˜25% of initial activity.

TABLE 13 Remaining Activity (% ± SD) Catalase-loaded Time (hours)Nanocarrier Free Catalase 0 100.0 ± 0.0  100.0 ± 0.0  0.5 83.4 ± 8.425.3 ± 0.2  1.0  91.7 ± 11.8 18.6 ± 3.9  2.0 65.6 ± 6.1 7.0 ± 2.8 4.048.5 ± 4.4 3.7 ± 1.5 6.0 39.8 ± 6.0 2.6 ± 1.3 22.0 24.8 ± 8.3 1.2 ± 0.6

To illustrate targeting of a polymeric nanocarrier of the presentinvention to a particular cell type, anti-PECAM-1 antibodies wereconjugated to the surface of the polymeric nanocarrier utilizing aPEG-tethered biotin-streptavidin linkage (Wu & Pardridge (1999) Proc.Natl. Acad. Sci. USA 96(1):254-9; (Hansen, et al. (1995) Biochim.Biophys. Acta 1239(2):133-44). Amine-terminated diblock PEG-PLGAcopolymers were synthesized using an art-established direct conjugationprocedure, providing 90% conjugation efficiency as determined by ¹H-NMR.The tertiary amine catalyst was not necessary, since a primary amine PEGwas used. NHS-Biotin reacted in anhydrous conditions resulted inconjugation onto the primary amine end of PEG, as determined by FTIR,which presented the carbonyl stretch of the urea bond (1630cm⁻¹) inbiotin.

To avoid aggregation, antibody-streptavidin conjugates were employed.Streptavidin was modified with the heterobifunctional cross-linkersuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) tointroduce maleimide groups, while SH-groups were introduced onto theantibody molecule using N-succinimidyl-S-acetylthioacetate (SATA). Assuch, an antibody was linked to streptavidin, forminganti-PECAM-streptavidin or IgG-streptavidin. Introducing one sulfhydrylper IgG molecule prevented multivalent cross-linking and aggregation.

Single emulsion-polymeric nanocarriers were covered with 130antibodies/polymeric nanocarrier which is 55% of the theoretical maximumIgG coverage. With an increase in size (double emulsion-polymericnanocarrier, 420 nm), 1200 antibodies/polymeric nanocarrier could beachieved, which is 30% of the theoretical maximum. This result may bedue to the internal voids contained within polymeric nanocarriers andtherefore less biotin is available for surface coverage; or due to thelower radius of curvature which reduces the surface accessibility of therelatively large streptavidin-IgG conjugate.

Coupling capacity of polymeric nanocarriers was analyzed using themodular IgG-streptavidin in combination with structurally uniform solidcore nanoparticles (solid-polymeric nanocarriers) of PEG-PLGA with andwithout 15 mol % biotin-PEG-PLGA. ¹²⁵I-IgG-streptavidin conjugates boundspecifically to biotin-containing polymeric nanocarrier (177.5±9IgGs/polymeric nanocarrier), but not biotin-free polymeric nanocarriers(3.0±0.1 IgGs/polymeric nanocarrier). This translates into a 33.5±1.7%of maximum coverage (assuming the 8000 antibodies/μ² maximum coveragefor pure polystyrene beads). With subsequent centrifugations, there wasno measurable antibody on the surface of the polymeric nanocarrierwithout biotin and the biotin-polymeric nanocarrier contained asignificant fraction of the initially bound amount. The biotin labeledpolymeric nanocarrier lost a small fraction of radiolabel with eachcentrifugation step. However, the noticeable decrease in pellet sizewith each centrifugation is indicative of a decrease in particle lossrather than detachment of antibody conjugate. Further, biotin-polymericnanocarrier readily resuspended without significant aggregation. Theinitial size of the polymeric nanocarrier preparations was 120 nm, yetwith centrifugation and resuspension, both polymeric nanocarriers with(169.6±12 nm) and without biotin (143±3 nm) exhibited a size increase.The difference in size (Δ26 nm) of these preparations was only slightlylarger than the expected increase in size as a result of antibodycoating (assuming a r_(h) of ˜10 nm). This difference may be a result inthe larger size of the antibody streptavidin-conjugate compared tounconjugated IgG.

Double emulsion nanocarrier-encapsulated catalase was also synthesizedwith 15 mol % biotin-PEG-PLGA. ¹²⁵I-IgG-streptavidin, but not control¹²⁵I-IgG (i.e., streptavidin-free) coated the nanocarrier containingcatalase with a high yield (1245±64 versus 46±34, respectively). Thus,total amount of IgG per nanocarrier containing catalase was higher thanthat for solid-polymeric nanocarrier, likely due to the larger size(˜420 nm versus 120 nm) of the nanocarrier containing catalase. However,surface coating of IgG was 28±1.4% of the theoretical max, confirmingthe general nature of this modular coupling strategy.

Polymeric nanocarrier binding to cells expressing PECAM was tested invitro. As a control for both cell specificity and adhesion specificity,blank and anti-PECAM-single emulsion-polymeric nanocarrier wereincubated (340 pM) for 1 hour with REN and REN/PECAM cells to visualizepolymeric nanocarrier binding. Anti-CAM targeting of latex beads resultsin localization at cell-cell borders (for anti-PECAM) and perinuclearregions as a result of internalization (Muro, et al. (2003) J. Cell Sci.116 (Pt 8) :1599-609). A similar pattern of localization was found withthe anti-PECAM/polymeric nanocarrier incubated with REN/PECAM,indicating that single emulsion-polymeric nanocarriers are capable ofbeing internalized by REN/PECAM cells. PECAM-single emulsion-polymericnanocarriers bound specifically to the REN/PECAM cells (117±11 polymericnanocarriers/cell), compared to wild-type REN cells (17.56±2 polymericnanocarriers/cell). Pure PEG-PLGA polymeric nanocarrier possessed littleto no binding to both REN/PECAM (2.2±0.5 polymeric nanocarriers/cell)and wild-type REN cells (0.0±0.0 polymeric nanocarriers/cell).

To obtain a more quantitative analysis of binding, polymericnanocarriers were partially labeled (5 mole % of total surface coatedconjugate) with a nonspecific ¹²⁵I-IgG-streptavidin. Particle bindingwas traced in an endothelial cell line (HUVEC) withIgG-streptavidin-coated polymeric nanocarrier as a control. It was foundthat anti-PECAM particle binding was dose-dependant with (193±1.5polymeric nanocarriers/cell) at a polymeric nanocarrier concentration of29 pM. When repeated with double emulsion-polymeric nanocarrier, bindingwas (330±101 polymeric nanocarrier/cell) at 22 pM. Due to the highstandard deviation of the double emulsion results, there was not astatistically significant difference between the single emulsion anddouble emulsion polymeric nanocarriers. In both experiments, IgG coatedparticles possessed little binding (15±3.5 and 15±0.7 polymericnanocarriers/cell for single emulsion and double emulsion formulations,respectively). These values are in close agreement with latex beadstudies, which have shown maximum binding to be ˜250 polymericnanocarriers/cell for 100 nm beads. Further, there appeared to bemaximum binding of polymeric nanocarrier reached for the doubleemulsion-polymeric nanocarrier but not for the single emulsion-polymericnanocarrier. This was likely a result of the greater size of the doubleemulsion-polymeric nanocarrier versus single emulsion-polymericnanocarrier, where it has been demonstrated that efficiency of CAMtargeting of submicron particles is distinctly size-dependant.

Toxicity, circulation and targeting in vivo were also performed.¹²⁵I-catalase-loaded polymeric nanocarriers (composed purely offunctionalized PEG-PLGA) were administered via i.v. to mice and ratswith no detectable pathological changes in the animals. Approximately70% of the injected dose of free catalase was cleared within 10 minutes,in agreement with published data (Muzykantov, et al. (1996) Proc. Natl.Acad. Sci. USA 93(11):5213-8). In contrast, catalase-loaded polymericnanocarrier circulated for a dramatically longer time in both rats andmice, with at least 60% of the injected dose present at 3 hours. Theinitial, more rapid, clearance of catalase-loaded polymeric nanocarrierduring the first 30 minutes may have been due to heterogeneities inpolymeric nanocarrier size which occur during formulation, alteredvasoreactivity in response to blood withdraw, or loss of surface boundfraction of catalase. The secondary clearance phase had a projectedhalf-life of 12 hours. Organ distribution was typical for sub 500 nmpolymeric nanocarriers. Liver and spleen displayed major uptake ofpolymeric nanocarrier containing catalase (although a significantfraction of catalase could be associated with the residual blood inthese highly perfused organs). There was no significant pulmonary uptakeof polymeric nanocarrier containing catalase, indicating lack ofintravascular aggregation and sequestration in the capillaries. Lack ofnon-specific or mechanical (embolization) pulmonary uptake was useful asa low baseline for targeting toward this vascular bed.

During the course of this analysis, more than one preparation ofpolymeric nanocarrier was employed. Plotting of the cumulative datacollected 1-hour post-injection revealed dependence of blood circulationon polymeric nanocarrier size. Even within a relatively narrow range of200-350 nm, circulation was reduced with increase in size. Therefore,modulation of polymeric nanocarrier size may provide for optimization ofdelivery and treatment design.

In similar in vivo analyses carried out with nanocarriers coated withanti-PECAM as an affinity moiety, ˜10% of the injected targetedpolymeric nanocarrier loaded with catalase was found in the lungs versus1% of the non-targeted catalase-loaded polymeric nanoparticles at 1 hourafter injection in mice. Furthermore, the blood distribution wasmarkedly reduced. Lung/blood ratio for anti-PECAM/polymericnanocarrier-catalase versus non-targeted polymeric nanocarriercontaining catalase was 2.7 versus 0.24, respectively. Thus, the ratioof lung uptake for targeted versus non-targeted polymeric nanocarriercontaining catalase normalized per blood level was above 10.

Hydrogen peroxide injury studies were performed to evaluate thetherapeutic potential of the anti-PECAM-targeted polymeric nanocarriers.Release of ⁵¹CrO₄ from pre-labeled cells was used as a marker ofcellular necrosis. Percent protection was defined by the followingequation (3),

$\begin{matrix}{P = {100 - {100*\frac{( {{Cr} - {Cr}_{0}} )}{( {{Cr}_{+} - {Cr}_{0}} )}}}} & (3)\end{matrix}$where P is percent protection, Cr is the chromium release of the sample,Cr₀ is the chromium release of uninjured (H₂O₂ null) cells and Cr₊ isthe chromium release of unprotected H₂O₂ injured cells.

For polymeric nanocarrier-protein-based therapy, a sufficient amount ofactive protein must be loaded and delivered to the site of injury toexert a benefit. In protection studies based upon ¹²⁵I-catalase tracingof catalase loaded polymeric nanocarriers, protection correlated withdelivered mass of drug (19.2±10.5% and 56.9±2.9 % protection for 7.0±1.5and 32±4.4 ng catalase/well, respectively). This result demonstratedthat even when relatively low amount of protein was delivered, amarginal amount of cellular protection was possible. When, carriers weretargeted, a greater amount of polymeric nanocarrier was present andprotection increased in response. Due to the small size of polymericnanocarriers, and therefore limited cargo capacity, initial activity ofthe loaded catalase greatly influenced the protection capabilities ofthe delivery system, with 56.9±2.9% and 100±8.6 % protection for the 30kU/mg- and 55 kU/mg-loaded catalase, respectively.

Using 55 kU/mg catalase-loaded polymeric nanocarriers,anti-PECAM-polymeric nanocarriers were pre-incubated for set times priorto injury to test the duration of therapeutic protection. Hydrogenperoxide concentration in the supernatant was measured to directlymonitor delivered catalase activity. From this monitoring of H₂O₂degradation and cellular protection, it was found that catalase loadedinto targeted polymeric nanocarriers was active and protected cells fortimes much longer than previously observed (up to 21 hours). Whilelittle visible change was noted in the first three hours, there was astatistically significant difference between concentrations of H₂O₂ at 5minutes. To track changes in effectiveness of catalase activity, a firstorder degradation model was fitted to each curve, where the timeconstant is equivalent to the total activity in the well. Normalizingthis residual activity with the initial values, a loss of catalyticactivity as a function of time was obtained. For pure protein conjugates(e.g., as described by Sweitzer, et al. (2003) supra; Kozower, et al.(2003) supra and Christofidou-Solomidou, et al. (2003) supra), activitywas almost completely lost by 2-3 hours of incubation with cells.Unexpectedly, the loss of delivered catalase activity mirrored the slowloss of activity in catalase-loaded polymeric nanocarriers observedunder artificial proteolytic conditions as disclosed herein. This resultindicates that the rate-limiting step in the loss of activity ofcatalase delivered to the cells may be controlled by the polymericnanocarrier, and therefore loss could be determined by varying theproperties of the delivering vehicle.

When catalase-loaded polymeric nanocarriers were incubated with cellsfor less than 3 hours (where activity was decreased by 30%) prior toinsult, enough active catalase was present to degrade 100% of thehydrogen peroxide in 10 minutes, translating into 100% cellularprotection for the first three hours. Further, catalase-loaded polymericnanocarriers incubated with cells for 24 hours possessed a stable 20% ofits initial catalase activity, which was capable of detoxifying hydrogenperoxide in 50 minutes, resulting in 56.7±8.7% protection. This level ofdelivery and capacity to protect against strong oxidizing conditionsindicates an unprecedented window of protection from a single, targetedcatalase dose. The overall protection potency (defined by the actual %protection divided by the 1 hour protection level), decreased at aslower rate than the actual loss of catalase activity. This resultdemonstrates that when a sufficient amount of catalase is present, adecoupling of protection from enzyme loss is possible, thereby providingeven greater activity durations than what would be otherwise possible.For instance, by adding twice as much needed catalase for protection,100% can be achieved for 8 hours (activity half-life ˜4 hours), whilefor pure protein conjugates such a load would only provide protectionfor <1 hour.

Therefore, targeted delivery of detoxifying enzymes encapsulated into asmall molecule-permeable polymer carrier of sub-micron size affordsprotection against damage from toxic small molecules for a duration oftime which has not been achieved by a single bolus of unencapsulatedenzyme. Thus, detoxifying enzymes in polymeric nanocarriers, which donot release the enzyme cargo, provides prolonged therapeutic actionintracellularly for use in methods for decreasing vascular oxidativestress and detoxifying xenobiotics.

Accordingly, the present invention is a method for decomposing areactive oxygen species (ROS) using a polymeric nanocarrier-encapsulatedantioxidant enzyme. The method involves contacting a sample containing aROS with the nanocarrier-encapsulated antioxidant enzyme so thatdecomposition of ROS to non-toxic low levels is achieved. A sample isintended to include, a biological sample (e.g., an organ fortransplantation), as well as an environmental sample or laboratorysample. For example, storage of organs for re-implantation is severelylimited by the ROS generated upon reperfusion of the transplanted organ.The ROS stress generated from reperfusion is proportional to the time ofischemia (storage time) inflicted upon the organ. By injecting thedonors before, or infusing organs immediately after the harvest withantioxidant nanocarriers, the “shelf-life of the organ can be greatlyextended, increasing the number of patients that can receivetransplants. Moreover, it is contemplated that ananocarrier-encapsulated antioxidant enzyme could be used to scavengeROS in chemical synthesis reactions.

Local administration of antioxidant enzymes or antioxidantenzyme-encoding genes, as well as antioxidant enzyme overexpression bytransgene technologies, has been shown to protect against oxidativestress in animal models (Fridovich (1995) Annu. Rev. Biochem. 64:97-112;Erzurum, et al. (1993) J. Appl. Physiol. 75(3) :1256-62). However,containment of vascular oxidative stress using exogenous antioxidantenzymes requires extended activity of the enzyme because the majority ofpathological conditions involving vascular stress take time ranging fromdays to weeks (e.g., inflammation, acute lung injury, stroke, hyperoxia,myocardial infraction and post-ischemic syndrome) and months to years(e.g., atherosclerosis, hypertension, diabetes). Thus, delivery of anantioxidant enzyme in a polymeric nanocarrier as disclosed hereinprovides prolonged therapeutic effect due to protection fromproteolysis. Moreover, when the instant polymeric nanocarrier containsan antioxidant enzyme and an affinity moiety for targeting vascularendothelial cells (e.g., anti-PECAM or anti-ICAM), an increase inefficacy of reducing vascular oxidative stress at vascular epitheliumcan be achieved. To effect protection against vascular oxidative stress,a subject in need of treatment (e.g., having pathological conditionswhich induce production of toxic oxidants) is administered an effectiveamount of a polymeric nanocarrier-encapsulated antioxidant compositionof the present invention. Such treatment desirably provides a detectabledecrease (i.e., detectable by either chemical methods documentingreduction of ROS levels or by biological outcomes such as survival orextent of organ damage) in the vascular oxidative stress in the subject.

Administration of the instant antioxidant enzyme compositions can beprior to medical procedures (e.g., surgery or radiation treatment) thatare known to cause ROS generation, thereby preventing the onset ofvascular oxidative stress and improving patient recovery, reducingrecovery time and hospitalization costs. Alternatively, administrationof the nanocarrier-encapsulated antioxidant enzyme can be as inintervention in debilitating situations such as acute lung injury,sepsis (toxic shock), autoimmune diseases, etc., thereby limiting theprogressive damage caused by ROS under these extreme oxidative stresssituations.

Differing administration vehicles, dosages, and routes of administrationcan be determined for optimal administration of the instant nanocarriercompositions; for example, injection near the site of an injury or tumormay be preferable for facilitating local treatment. For example,biodegradable nanocarriers composed of PLGA with anti-inflammatory(e.g., hydrocortisone) and growth factors (e.g., BDNF) encapsulatedtherein can be administered via direct lumbar injection using a standardspinal tap procedure. Nanocarriers introduced into the cerebral spinalfluid are dispersed through this space via natural convective motion andaccumulate at the wound site as a result of the enhanced permeation andretention (EPR) effect (Torchilin (2000) Eur. J. Pharm. Sci. 11 (Suppl.2):S81-91). Also, targeting can be further enhanced by the inclusionantibodies toward common inflammatory markers.

Generally, the nanocarrier compositions used in the invention areadministered to an animal in an effective amount. Generally, aneffective amount is an amount of encapsulated protein effective toeither reduce the symptoms of the disease sought to be treated or inducea pharmacological change relevant to treating the disease sought to betreated. Therapeutically effective amounts of the encapsulated proteinscan be any amount or doses sufficient to bring about the desired effectand depend, in part, on the condition, type and location of thepathology, the size and condition of the patient, as well as otherfactors readily known to those skilled in the art. The dosages can begiven as a single dose, or as several doses, for example, divided overthe course of several weeks.

The polymeric nanocarrier compositions of the instant invention can beadministered by any suitable means, including, e.g., parenteral,topical, oral or local administration, such as by injection (e.g., intothe vasculature) or by aerosol (e.g., into the lungs). In certainembodiments, administration is by injection. Such injection can belocally administered to any affected area. For particular modes ofdelivery, a polymeric nanocarrier composition of the instant inventioncan be formulated with an excipient. See, e.g., Remington: The Scienceand Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed.Lippincott Williams & Wilkins: Philadelphia, Pa., 2000. Routes ofadministration of targeted polymeric nanocarriers can be by intravenous,interperitoneal, or subcutaneous injection including administration toveins or the lymphatic system. While the primary focus of the inventionis on vascular-targeted nanocarriers, in principle, a targetednanocarrier can be designed to focus on markers present in other fluids,body tissues, and body cavities, e.g., synovial fluid, ocular fluid, orspinal fluid. Thus, for example, a nanocarrier can be administered tospinal fluid, where an antibody targets a site of pathology accessiblefrom the spinal fluid. Intrathecal delivery, that is, administrationinto the cerebrospinal fluid bathing the spinal cord and brain, may beappropriate for example, in the case of a target residing in the choroidplexus endothelium of the cerebral spinal fluid (CSF)-blood barrier.Polymeric nanocarrier compositions can be administered to any animal,desirably to mammals, and more desirably to humans.

In addition to ROS, the instant invention embraces the detoxification ofxenobiotics. As disclosed herein, xenobiotics encompass a variety ofagents which can be detoxified by, e.g., peroxisomal enzymes. Xenobioticdetoxification can be carried out in vivo or in vitro. As such, onemethod of the present invention involves contacting a sample containinga xenobiotic with a polymeric nanocarrier-encapsulated detoxifyingenzyme composition so that the xenobiotic is detoxified. In the contextof this method, a sample can be of biological or environmental origin,or any other source contaminated with a xenobiotic. For example, it iscontemplated that a polymeric nanocarrier-encapsulated dehalogenasecould find application in environmental remediation.

In accordance with in vivo applications, a polymericnanocarrier-encapsulated detoxifying enzyme is administered to a subjectexposed to a xenobiotic in amount which effectively detoxifies thexenobiotic. Generally, an effective amount is an amount of encapsulatedprotein effective to either reduce the symptoms associated with thexenobiotic or detoxify the xenobiotic present in the subject to lesstoxic or non-toxic levels. For example, by injecting the instantnanocarrier composition into the vascular endothelium, which is directlyexposed to circulating toxins, xenobiotics can be rapidly degraded(oxidized) into more inert, water-soluble forms, allowing for a morerapid recovery time. Such a strategy would allow for a benign and moregenerically applicable first treatment of unknown or antidote-lackingtoxins and allow for prophylaxis of at risk populations (e.g.,accidental industrial chemical release or military personnel in achemical warfare setting).

As will be appreciated by the skilled artisan, in addition to loadinglarge proteins (e.g., catalase having a molecular weight of 240 kD), theinstant method of preparing polymeric nanocarriers can be used forloading small to middle-size proteins without enzymatic activity (e.g.,insulin or albumin) or other molecules which are sensitive toshear-induced deactivation during polymer emulsification (e.g., nucleicacids, carbohydrates, organic compounds, etc).

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 Materials

All reagents were used as received unless indicated otherwise.Methoxypoly(ethylene glycol) molecular weight 5000 (mPEG) was purchasedfrom Polysciences (Warrington, Pa.). Poly(lactic-co-glycolic acid)(50:50) in the free acid (38,000 molecular weight) form was purchasedfrom ALKERMES®, Inc. (Cincinnati, Ohio). Bovine liver catalase (242,000Dalton) was obtained from CALBIOCHEM® (EMD Biosciences, San Diego,Calif.). 10-acetyl-3,7-dihydroxyphenoxazine (AMPLEX® Red) and ALEXAFLOUR®-488 goat anti-mouse antibodies were purchased from MOLECULARPROBES™ (Eugene, Oreg.). Succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC);N-succinimidyl-S-acetylthioacetate (SATA); and N-succinimidyl-biotin(NHS-biotin) were obtained from Pierce Biotechnology (Rockford, Ill.).Na¹²⁵I and Cr⁵¹ were purchased from PERKINELMER™ (Boston, Mass.). Allother reagents and solvents were obtained from Sigma-Aldrich (St. Louis,Mo.).

EXAMPLE 2 Synthesis of Diblock Copolymers

Diblock copolymers were prepared by three different methods.

PEG-PLGA. PLGA (50:50) polymer containing a carboxylate end group andPEG (10,000 molecular weight) was freeze-dried overnight to remove boundwater. The polymers were mixed in a 4:1 molar ratio (PEG/PLGA 38 kDa) inanhydrous dichloromethane (DCM) to a final polymer concentration of 10weight %. Subsequently, 2,2-dicyclocarbodiimide (DCC) andN,N′-dimethylaminopyridine (DMAP) were added to the PLGA at the molarratio of 4:1:2. Conjugation was carried out under a N₂ atmosphere atroom temperature for 18 hours. The resulting dicyclohexylureaprecipitate was filtered out and the polymer was precipitated twice inanhydrous ether. The filtrate was dried and dissolved in cold acetone.The insoluble fraction was filtered out and once more precipitated incold ethanol.

mPEG-PLA. DL-lactide was recrystallized twice in anhydrous ether, andmixed with mPEG in weight ratios predetermining their molecular weight(30 kDa). The bulk material was raised to 140° C. for 2 hours under areduced nitrogen atmosphere. Subsequently, the temperature was reducedto 120° C., 1 weight % stannous 2-ethyl-hexanoate was added, and thepolymerization proceeded for 6 hours. The resulting polymer wasdissolved in dichloromethane (DCM), and precipitated twice in colddiethyl ether. The final product was serially dried in a rotovap (SafetyVap 205; Buchi, Switzerland) and a freeze dryer (RCT 60; Jouan, Inc.,Winchester, Va.) to remove any residual solvent.

Biotin-PEG-PLGA. PLGA (50:50; 38 kDa) polymer containing a carboxylateend group and PEG-Diamine (10,000 molecular weight) was freeze-driedovernight to remove bound water. The polymers were mixed in a 6:1 molarratio (PEG:PLGA) in anhydrous DCM to a final polymer concentration of 2weight %. Subsequently, DCC was added to at the molar ratio of 1.2:1(DCC:PLGA). Conjugation was carried out under a N₂ atmosphere at roomtemperature for 18 hours. The resulting dicyclohexylurea precipitate wasfiltered out, and the polymer was precipitated twice in anhydrous ether.The filtrate was then dried, dissolved in acetone and precipitated indeionized water. The precipitate was filtered and freeze-dried.Subsequently, biotin-N′-succinimidyl ester was added (1.2:1 molar ratio)with the polymer in DCM. After 4 hours, the polymer was precipitatedtwice in ether.

The chemistry of the polymer was verified by FTIR (Nicolet Magna IR560;Thermo Nicolet Corp., Madison, Wis.), gel permeation chromatography(GPC) using 2 serial pLGel Mixed C columns 300×7.5 mm (PolymerLaboratories, Amherst, Mass.) with an Acuflow Series III pump and aDifferential Refractometer (Knauer, Berlin, Germany) calibrated usingpolystyrene standards to evaluate polymer molecular weight and the PDI.Relative PEG content was determined as disclosed herein.

EXAMPLE 3 PEG and PLA Content Determination

A 50 μL aliquot of the concentrated nanocarrier prep was saponified byadding 200 μL of 5 M NaOH and reacting the mixture overnight at 80° C.The solution was neutralized with 200 μL of 5 M HCl. PEG concentrationwas determined by a colorimetric assay based upon a PEG-Barium Iodidecomplex. Absorbance of the color product was measured at 550 nm using amicroplate reader (Model 2550-UV; BIO-RAD® Labs, Hercules, Calif.) (Simsand Snape (1980) Anal. Biochem. 107:60-63).

To measure PLA concentration, an enzymatic assay for L-lactic acid wasused. 5 μL of sample was added to 45 μL of 50 mM PBS in a microplatewell. To this well was added 50 μL of assay buffer. The assay bufferconsisted of 2 U/mL horseradish peroxidase, 20 mU lactate oxidase and 1μg/mL of AMPLEX® Red. After a 10-minute incubation at room temperature,the resorufin product concentration was determined by UV absorbance at550 nm. Concentrations were measured in triplicate for each individualparticle preparation.

EXAMPLE 4 Determination of H₂O₂ Diffusivity in PLGA

The diffusivity of H₂O₂ through PLGA was determined by using atwo-chamber diffusion apparatus. Polymer films of esterified PLGA(34,000 molecular weight) were prepared via solvent casting procedure.The donor cell contained a 5 mM H₂O₂ solution in phosphate-bufferedsaline (PBS; 50 mM, 7.4 pH), and the receptor cell contained pure PBSbuffer. At specific time intervals (15 and 30 minutes), the receptorcell contents were removed and replaced with fresh buffer. Theconcentration of the H₂O₂ in the receptor cell was determined by UVabsorbance at 242 nm (Cary 50 UV-Vis; VARIAN® Inc., Palo Alto, Calif.).Diffusivity studies were performed in triplicate for two independentlycast polymer films.

EXAMPLE 5 Nanoparticle Synthesis

Two types of polymeric nanocarrier syntheses were employed, non-catalaseloadable single-emulsion polymeric nanocarrier, and catalase in a doubleemulsion polymeric nanocarrier.

Single-Emulsion Polymeric Nanocarrier. PEG-PLGA with and without 15 mol% biotin-PEG-PLGA was dissolved in acetone (10 mg/mL, 2.5 mL). Thissolution was slowly pipetted into 20 mL PBS under mild agitation.Acetone was removed under vacuum using a dry nitrogen stream.Single-emulsion polymeric nanocarrier was collected by centrifugation at30,000 g for 30 minutes. The pellet was resuspended in 1 mL of PBS.Stock concentrations were determined according to chemical and enzymaticassay.

Double Emulsion Polymeric Nanocarrier. A primary emulsion was formed byhomogenizing at 15 krpm for 1 minute (−80° C., dry ice/acetone bath) a100 μL aqueous drug solution (1-25 mg/mL catalase in PBS) in a 1 mLorganic polymer solution (25 mg/mL PEG-PLGA in DMC or for someapplications mPEG-PLA with 15 mol % biotin-PEG-PLA in DCM), using a 7-mmblade homogenizer (Kinematica POLYTRON® 3100 equipped with a PTDA3007/2generator; Brinkmann Instruments, Westbury, N.Y.). This primary emulsionwas immediately pipetted into a secondary aqueous phase (5 mL)containing 2 weight % poly(vinyl alcohol) (PVA; 10,000 molecular weight,80% hydrolyzed), sodium cholate, or 2 weight % PLUORNIC™ (F68) andhomogenized at 15 krpm for 1 minute. This second homogenization wasadded to an additional 10 mL of the same surfactant solution, andstirred overnight at room temperature under mild agitation to removeresidual solvent.

To purify the resultant nanoparticles, a serial centrifugation schemewas used. The solution was first centrifuged at 1000 g for 15 minutes toremove the large microparticle/macroaggregate fraction. The supernatantwas then centrifuged at 22,000 g for 30 minutes. Nanoparticles wererinsed twice more to remove residual surfactant and unloaded protein.Alternatively, to select for nanocarriers of 100 to 300 nm, nanocarrierswere filtered through a 1 μm filter. All preparations were performed intriplicate. Particle sizes were determined by dynamic light scattering(0 PLUS Particle Sizer; Brookhaven Instruments, Holtsville, N.Y.).

EXAMPLE 6 Antibody-Streptavidin Conjugate Preparation

Heterobifunctional cross-linker succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) was used tointroduce stable maleimide reactive group into streptavidin molecules.The reaction was performed at 40-fold molar excess of SMCC at roomtemperature for 1 hour. In parallel, sulfhydryls were introduced in theantibody or control IgG through primary amine usingN-succinimidyl-S-acetylthioacetate (SATA). The yield of the reaction wasabout 20%. Thus, to introduce 1 sulfhydryl per IgG molecule, antibodywas incubated with 5-fold molar excess of SATA at room temperature for30 minutes. This extent of modification prevented possible cross-linkingof streptavidin and subsequent protein polymerization. Sulfhydryls weredeprotected using hydroxylamine and antibody was conjugated withstreptavidin at 2:1 IgG to streptavidin molar ratio. At each stepunreacted components were removed using Spin Protein Columns (G-25SEPHADEX™; Roche Applied Science, Indianapolis, Ind.).

EXAMPLE 7 Analysis of Enzymatic Activity

The activity of catalase was determined using a standard catalase assay(Shuvaev, et al. (2004) Methods Mol. Biol. 283:3-20). Briefly, 900 or950 μL of a 5 mM H₂O₂ solution in PBS (7.4 pH) was added to a quartzcuvette at ambient conditions. A catalase-loaded nanoparticle solutionwas added to bring the total volume to 1 mL. The concentration of H₂O₂was monitored versus time by measuring the absorbance at 242 nm (1Unit=23.0 ·[Δabsorbance/min]). The activity was measured twice at twodifferent concentrations (50 and 100 μL) for each individual particlepreparation.

EXAMPLE 8 Loading Analysis

Loading analysis was indirectly calculated by measuring the¹²⁵I-catalase content in solution pre- and post-centrifugations (n=3).Protein content was determined by radiotracing using a WIZARD® 1470gamma counter (Wallac Oy, Turku, Finland). Catalase was radiolabeledwith Na¹²⁵I (PERKINELMER™, Boston, Mass.) using the IODOGEN® (PierceBiotech., Rockford, Ill.) method, and unbound iodine was removed fromprotein using gel permeation chromatography (Biospin 6 Columns, BIO-RAD®Labs, Hercules, Calif.). Conditions were based upon manufacturer'srecommendations.

EXAMPLE 9 Determination of Protection of Enzyme

To evaluate the ability of nanoparticles to protect the activity ofloaded enzyme, an in vitro proteolytic assay was employed. In thesestudies, nanoparticles were incubated at 37° C. in a PBS solutioncontaining 0.2 weight % PRONASE®, a robust proteolytic enzyme. Aliquotswere taken at specific intervals of incubation and measured for eitherenzymatic activity or protein loading content.

EXAMPLE 10 Cell culture

Pooled human umbilical vein endothelial cells, HUVEC (CLONETICS®, SanDiego, Calif.), were cultured at 37° C., 5% CO₂, and 95% relativehumidity in supplemented M199 medium (GIBCO BRL™, Grand Island, N.Y.)and used at passage 4-5. Non-endothelial REN cells (human mesothelioma)were maintained in RPMI 1640 medium supplemented with 10% fetal bovineserum (HYCLONE®, Logan, Utah), 2 mM glutamine, 100 U/mL penicillin, and100 μg/mL streptomycin. REN cells stably transfected with human PECAM(REN/PECAM cells, which express PECAM-1 at levels and cellularlocalization similar to those found in human endothelial cells weremaintained in the same growth medium supplemented with 0.5 mg/mLG418-sulfate. G418-sulfate was omitted from the medium duringexperiments. For microscopy studies, cells were seeded onto 12-mmcoverslips in 24-well plates. For all other studies, cells were coateddirectly onto 24-well plates. To ensure attachment, HUVEC cells wereseeded only onto gelatinized surfaces.

EXAMPLE 11 Binding Studies

Binding studies were carried out using both radiolabeling andfluorescent microscopy. Fluorescence studies were carried out using RENand REN/PECAM cells mounted onto glass coverslips. Cells were incubatedwith polymeric nanocarriers (single-emulsion polymeric nanocarrier andanti-PECAM-single-emulsion polymeric nanocarrier) for 1 hour at 37° C.Cells were then washed 5 times with PBS and fixed (2% paraformaldyhde,15 minutes ambient conditions). Cells were washed and labeled with agreen fluorescent antibody. Coverslips were then mounted and imaged.Particle binding was semi-quantitatively determined by image analysis.

For Radiotracing studies, polymeric nanocarriers (IgG-polymericnanocarrier and anti-PECAM-polymeric nanocarrier) were labeled with¹²⁵I-IgG-streptavidin conjugate (5% of total conjugate surface coating).Cells were incubated for 1 hour at 37° C., washed 5 times, and lysedusing 1% TRITON™ X-100 in 1N NaOH. Cell supernatants and lysates werecollected and counted to determine extent of binding.

EXAMPLE 12 Protection from Cell Injury by H₂O₂

Cellular injury in culture was determined by the specific release of⁵¹CrO₄. To label the HUVEC cells, ⁵¹Cr isotope (200,000 cpm/well) wasadded 24 hours prior to the experiment. The HUVEC were washed andincubated with double emulsion-polymeric nanocarrier (anti-PECAM and IgGlabeled) for 1 hour in HUVEC medium. Cells were then washed 5 times withRPMI 1640 without phenol red. H₂O₂ (5 mM) was added to the cells and thecells were incubated for 5 hours at 37° C. with 5% CO₂. Totalradioactivity in the supernatant and in the cell lysates was determined.

At the indicated times, H₂O₂ remaining in the supernatant medium wasmeasured by H₂O₂-dependent oxidation of o-phenylenediamine (15 mM finalconcentration) in the presence of horseradish peroxidase (5 μg/mL finalconcentration) as determined by absorbance at 490 nm in a BIO-RAD® 3550Microtiter Plate Reader.

Stability of catalase loaded-polymeric nanocarriers was determined byincubating polymeric nanocarriers with cells for various times prior toH₂O₂ insult. During the 24 period of ⁵¹CrO₄ incubation, doubleemulsion-polymeric nanocarrier was introduced into thechromium-containing medium at specific times for 1 hour, the cells werewashed 5 times with HUVEC medium, and incubated with chromium-containingmedium for the remainder of the 24-hour period. Cells were subsequentlywashed and injury was assessed as described herein.

1. A method for producing a polymeric carrier encapsulated proteincomposition comprising: (a) homogenizing an aqueous solution of at leastone protein, with an amphiphilic polymer in an organic solvent at afirst temperature below 0° C. so that a first emulsion is produced; (b)homogenizing the first emulsion with an aqueous phase containing astabilizing surfactant at a temperature of between 4° C. to 25° C.; and(c) removing the organic solvent; wherein a polymericcarrier-encapsulated protein composition is produced.
 2. The method ofclaim 1, wherein the polymer is an amphiphilic diblock copolymer.
 3. Themethod of claim 1, wherein said first temperature comprises atemperature in the range of −180° C. to 0° C.
 4. The method of claim 1,wherein the protein is an enzyme.
 5. The method of claim 1, wherein saidfirst temperature comprises a temperature between the freezing point ofthe organic solvent and the melting point of the protein.
 6. The methodof claim 1, wherein the organic phase is removed by evaporation.
 7. Themethod of claim 1, wherein the polymer has a functional group forphysically cross-linking with an affinity moiety.
 8. The method of claim1, wherein the resulting polymeric carrier composition is of a sizebetween 20 nm−20 microns and is permeable to the substrate of theencapsulated protein, protects the protein from protease degradation,and preserves protein activity.
 9. The method of claim 2, wherein theamphiphilic diblock copolymer comprises PEG-PLGA.
 10. The method ofclaim 3, wherein said first temperature comprises a temperature in therange of −100° C. to −40° C.
 11. The method of claim 4, wherein theprotein is an antioxidant enzyme.
 12. The method of claim 4, wherein theprotein is a detoxifying enzyme.
 13. The method of claim 4, wherein theprotein is an enzyme with a molecular weight up to 240 kD.
 14. Themethod of claim 8, wherein the resulting polymeric carrier compositionis in the size range of 50 to 500 nm.
 15. The method of claim 13,further comprising the step of conjugating an affinity moiety to thesurface of the carrier.
 16. A method for producing a polymeric carrierencapsulated protein composition comprising: (a) homogenizing an aqueoussolution of at least one protein, with an amphiphilic polymer having afunctional group for physically cross-linking with an additionalmolecule in an organic solvent at a first temperature below 0° C. sothat a first emulsion is produced; (b) homogenizing the first emulsionwith an aqueous phase containing a stabilizing surfactant at atemperature of between 4° C. to 25° C.; (c) removing the organic solventwherein a polymeric carrier-encapsulated protein composition isproduced; and (d) conjugating to the functional groups an additionalmolecule.
 17. The method of claim 16, wherein the polymericcarrier-encapsulated protein composition is of a size between 20 nm−20microns and is permeable to the substrate of the encapsulated protein,protects the protein from protease degradation, and preserves proteinactivity.
 18. The method of claim 16, wherein the functional group isselected from the group consisting of: amines, hydroxyls, carbonyls,thiols, carboxylic acids and biotin.
 19. The method of claim 16, whereinthe additional molecule is a therapeutic molecule.
 20. The method ofclaim 16, wherein the additional molecule is a polymer.
 21. The methodof claim 16, wherein the additional molecule is an affinity moiety. 22.The method of claim 16, wherein the polymer is an amphiphilic diblockcopolymer.
 23. The method of claim 21, wherein the affinity moiety is anantibody or an antibody fragment.